Abstract Development and validation of a precision genome editing platform The development of a programmable way to achieve precision gene editing would represent both a powerful new research tool, as well as a potential new approach to gene editing-based human therapeutics. Current genome engineering tools suffer from modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration. This proposal seeks to engineer fusion proteins with readily programmable, site specific C>T DNA editing capabilities. The fusions will take advantage of the Cas9 system, an effector complex comprised of a Cas protein that is targeted to a specific DNA sequence by an RNA molecule (termed sgRNA). Fusion complexes between Cas9 and cytidine deaminase enzymes will be engineered in order to direct their ability to deaminate cytidine to uridine to a specific site in genomic DNA. Both the enzymatic activities and selectivities of the various simple fusions will be characterized using a luciferase-based assay in vitro. These results will be thoroughly analyzed and examined, as they may provide fundamental information about the relationship between the structure of Cas9 and its ability to access its target DNA. If the engineered fusions display little to no activity, or significant off-target effects, phage-assited continuous evolution (PACE), a state-of-the-art protein evolution method, will then be used to optimize the activities and specificities of the fusions. This method has been successfully used in the past to evolve mutant polymerases starting from undetectable activities. The resulting fusion enzymes will then be validated by correcting the cancer relevant H1047R (A3140G) mutation in the PIK3CA gene in a human cancer cell line. The successful development of these fusion enzymes would represent the initial phase of the development of a tool that can be used to investigate various biological problems. This technology could be used to knock out proteins at will with high efficiencies and observe the outcomes, study the effect of site specific protein mutations on signaling pathways and cell function, and investigate cancer progression following correction of a mutated gene.